Graft copolymers of polycationic species and water-soluble polymers, and use therefor

ABSTRACT

In accordance with the present invention, there are provided methods to render cells non-adhesive and/or non-immunogenic with respect to macromolecules typically encountered in culture media or in physiological media. The invention method comprises contacting cells with an effective amount of a composition comprising a polycationic species having water-soluble polymer chains grafted thereon.

The present invention relates to methods for rendering cellsnon-adhesive. In another aspect, the present invention relates tomethods for rendering cells non-immunogenic. In yet another aspect, thepresent invention relates to methods for the stabilization of liposomes.In a further aspect, the present invention relates to methods for the invitro generation of neural networks.

BACKGROUND OF THE INVENTION

Water-soluble polymers, such as polyethylene glycols (PEGs), have beeninvestigated extensively in recent years for use as biocompatible,protein repulsive, noninflammatory, and nonimmunogenic modifiers fordrugs, proteins, enzymes, and surfaces of implanted materials. Thesecharacteristics have variously been attributed to a combination ofproperties of such polymers, e.g., nonionic character, water solubility,backbone flexibility, and volume exclusion effect in solution or whenimmobilized on a surface.

While extensive efforts have been made to render foreign substances,such as drugs, proteins, and the like, non-immunogenic employingwater-soluble polymers such as PEG, the use of such polymers to renderan individual cell non-immunogenic has not been considered in the art.If such polymers could be attached directly to a cell surface, then itis possible, due to the exclusion of proteins and macromolecules fromthe vicinity of the cell surface, that the cell as a whole may berendered non-immunogenic. The ability to accomplish such attachmentwould be invaluable for a variety of treatment protocols.

It is known that mammalian cell membranes have a variety of negativelycharged species on their exterior. For example, negatively chargedproteoglycans (PGA), glycosaminoglycans (GAG), such as chondroitinsulfate and heparin sulfate, and negatively charged lipids have all beenidentified on cell exteriors. Not surprisingly, polycation species suchas polylysine and polyornithine interact with negatively charged cellsurfaces to form a strong ionic linkage. Unfortunately, polycations(such as polylysine and polyornithine) are known to be cytotoxic, evenat fairly low concentrations. Polylysine, for example, has been used asa cell fixative, and has been shown to cause cell aggregation (e.g.,with human platelets).

While water-soluble polymers, having found use in a variety ofbiological applications, would be ideal for use in treating cells torender them non-immunogenic, their generally non-ionic nature rendersthem substantially unable to bind to cell membranes. Thus, for example,treatment of cells with unmodified PEG was unable to confer anon-adhesive or protein repellant character on such cells.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have developed methods torender cells non-adhesive and/or non-immunogenic with respect tomacromolecules typically encountered in culture media or inphysiological media.

The methods of the present invention can be used for a wide variety ofpurposes, e.g., for the treatment of cells used for implantation(thereby avoiding the need for immunosuppressive therapy), for thepreservation of organs outside the body while awaiting transplant, formodifying surfaces of materials which are to be exposed to variouscomponents of physiological media, for the stabilization of liposomes(and prevention of their uptake by the reticuloendothelial system), andthe like.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a method torender cells non-adhesive, said method comprising contacting said cellswith an effective amount of a composition comprising a polycationicspecies having water-soluble polymer chains grafted thereon.

As employed herein, reference to rendering cells "non-adhesive" means,in an in vitro setting, that cells do not stick to wells (e.g., glass,plastic, and the like), or other surfaces with which they come incontact. Instead, non-adhesive cells, as contemplated herein, spread andgrow, yet remain in suspension. In an in vivo setting, "non-adhesive"refers to cells which do not adhere to biologically-encounteredmacromolecules or proteinaceous matrix (e.g., collagen matrix). As usedherein, "non-adhesive" also refers to cells which have been renderednon-immunogenic, i.e., cells which are substantially non-susceptible toan immune response mediated by biological macromolecules.

"Contacting" of cells or tissues with graft copolymer compositionscontemplated for use in the practice of the present invention istypically carried out in vitro at room temperature for a time in therange of about 0.01 up to 1 hour or longer in suitable physiologicalbuffer (i.e., pH in the range of about 7.2-7.4; osmolarity of about 290mOsm/kg) containing a concentration of at least about 0.005% of graftcopolymer, with respect to the concentration of the polycationic speciesused for the preparation of the cell surface (e.g., polylysine). It ispresently preferred to treat cells with a solution of graft copolymercontaining a concentration of graft copolymer in the range of about 0.05up to 1.0%, with concentrations of graft copolymer in the range of about0.1 up to 0.5% being especially preferred. Those of skill in the artrecognize that as the molecular weight of the polycationic species isincreased, a lower concentration (determined on the same basis as setforth above) of the graft copolymer is required to produce the samebeneficial effect.

As employed herein, an "effective amount" of graft copolymercompositions contemplated for use in the practice of the presentinvention is an amount sufficient to render said cells non-adhesive tobiological macromolecules, while leaving the cells viable (asdetermined, for example, by suitable staining techniques). In the caseof specialized cells, such as islets, it is desirable for the treatedcells to retain their unique function as well as viability (i.e., theability of islets to respond to exposure to glucose by secretion ofinsulin). Typically, an excess of graft copolymer is used with respectto the negative charges present on the surface of the cells to betreated. The quantity of graft copolymer required will vary depending onthe cell type being treated and the concentration of cells to betreated. Typically, in the range of about 10² -10⁸ cells/ml will betreated. For example, up to about 10⁸ bacterial cells/ml, up to about100,000 fibroblasts/ml, or up to about 50,000 islets/ml will be treated.

Copolymer compositions contemplated for use in the practice of thepresent invention comprise a polycationic species having water-solublepolymer chains grafted thereon. Polycationic species contemplated foruse in the practice of the present invention are polycationic specieshaving sufficient charge density to allow binding of the above-describedgraft copolymer to cells, and include:

polymers containing primary amine groups, secondary amine groups,tertiary amine groups or pyridinyl nitrogen(s), such aspolyethyleneimine, polyallylamine, polyetheramine, polyvinylpyridine,and the like,

polysaccharides having a positively charged functionality thereon (e.g.,chitosan),

polyamino acids, such as:

poly-L-histidine, poly-im-benzyl-L-histidine,

poly-D-lysine, poly-DL-lysine, poly-L-lysine,

poly-ε-CBZ-D-lysine, poly-ε-CBZ-DL-lysine,

poly-ε-CBZ-L-lysine,

poly-DL-ornithine, poly-L-ornithine,

poly-δ-CBZ-DL-ornithine,

poly-L-arginine,

poly-DL-alanine-poly-L-lysine;

poly (-L-histidine, L-glutamic acid)-poly-DL-alanine-poly-L-lysine;

poly(L-phenylalanine, L-glutamic acid)-poly-DL-alanine-poly-L-lysine;

poly(L-tyrosine, L-glutamic acid)-poly-DL-alanine-poly-L-lysine;

random copolymers of:

L-arginine with tryptophan, tyrosine, or serine;

D-glutamic acid with D-lysine;

L-glutamic acid with lysine, ornithine, or mixtures thereof;

and the like, as well as mixtures of any two or more thereof.

Presently preferred polycations for use in the practice of the presentinvention include polylysine (i.e., poly-D-lysine (PDL), poly-DL-lysine,poly-L-lysine (PLL), poly-ε-CBZ-D-lysine, poly-ε-CBZ-DL-lysine, orpoly-ε-CBZ-L-lysine), polyornithine (i.e., poly-DL-ornithine,poly-L-ornithine or poly-δ-CBZ-DL-ornithine), and the like.

Polycationic species having a wide range of molecular weights can beemployed in the practice of the present invention. Polycations having aMW in the range of about 200 up to 1,000,000 are suitable, withmolecular weights in the range of about 1000 up to 100,000 preferred.Presently most preferred polycationic species for use in the practice ofthe present invention will have molecular weights in the range of about5,000 to 50,000.

Optionally, the polycationic species employed in the practice of thepresent invention can be further modified with one or more functionalgroups capable of undergoing free radical polymerization. Suitablefunctional groups for this purpose include unsaturated species capableof free radical polymerization, such as, for example, acrylate groups,vinyl groups, methacrylate groups, and the like. When cells or tissuesare treated with such modified polycationic species, the graft copolymercan be further subjected to free radical polymerization conditions,thereby stabilizing the association of graft copolymer with the cellsurface. In addition, the further crosslinking of the graft copolymerforms a highly stabilized, immunoprotective coating of water-solublepolymer about the treated cell or tissue.

Free radical polymerization of the above-described modified polycationicspecies can be carried out in a variety of ways, for example, initiatedby irradiation with suitable wavelength electromagnetic radiation (e.g.,visible or ultraviolet radiation) in the presence of a suitablephotoinitiator, and optionally, cocatalyst and/or comonomer.Alternatively, free radical polymerization can be initiated by thermaldecomposition of a suitable free radical catalyst, such as benzoylperoxide, azobisisobutyronitrile, and the like.

Photoinitiators contemplated for use in the practice of the presentinvention include such ultraviolet (UV) initiators as 2,2-dimethylphenoxyacetophenone, benzophenones and ionic derivatives (for watersolubility), benzils and ionic derivatives thereof, thioxanthones andionic derivatives thereof; and visible photoinitiators such as ethyleosin, eosin, erythrosin, rose bengal, thionine, methylene blue,riboflavin, and the like.

Cocatalysts are typically used when the excited state of thephotoinitiator is quenched too rapidly to efficiently promotepolymerization. In such a situation, a cocatalyst (also referred to inthe art as a "cosynergist", "activator", "initiating intermediate" or"quenching partner") will generally be employed. Cocatalystscontemplated for use in the practice of the present invention includetriethanolamine, methyl diethanolamine, triethylamine, arginine, and thelike.

Water-soluble polymeric species contemplated for use in the practice ofthe present invention are water-soluble polymers capable of renderingpolycations non-immunogenic and include non-ionic, water-solublepolymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA),poly(hydroxyethyl methacrylate) (pHEMA), poly(acrylamide), poly(vinylpyrrolidone) (PVP), poly(ethyl oxazoline), polysaccharides (such as, forexample, starch, glycogen, guar gum, locust bean gum, dextran, levan,inulin, cyclodextran, agarose, and the like); as well as ionic,water-soluble polymers such as polyacrylic acid (PAA) or polysaccharides(such as, for example, xanthan gum, carageenan, hyaluronic acid,heparin, chitosan, pectin, and the like); as well as copolymers of anytwo or more of said water-soluble polymeric species. Presently preferredwater soluble polymers employed in the practice of the present inventionare polyalkylene oxides, such as polyethylene glycol (PEG).

Water-soluble polymeric species having a wide range of molecular weightscan be employed in the practice of the present invention. Polymericspecies having a MW in the range of about 200 up to 1,000,000 aresuitable, with molecular weights in the range of about 500 up to 100,000preferred. Presently most preferred polymeric species for use in thepractice of the present invention will have molecular weights in therange of about 1000 to 50,000.

Optionally, the water-soluble polymeric species employed in the practiceof the present invention can be further modified with one or morefunctional groups capable of undergoing free radical polymerization.Suitable functional groups for this purpose include unsaturated speciescapable of free radical polymerization, such as, for example, acrylategroups, vinyl groups, methacrylate groups, and the like. When cells ortissues are treated with such modified water-soluble polymeric species,the graft copolymer can be further subjected to free radicalpolymerization conditions, thereby stabilizing the association of graftcopolymer with the cell surface. In addition, the further crosslinkingof the graft copolymer forms a highly stabilized, immunoprotectivecoating of water-soluble polymer about the treated cell or tissue.

Free radical polymerization of the above-described modifiedwater-soluble polymeric species can be carried out in the same manner asdescribed above with respect to free radical polymerization of modifiedpolycationic species.

Graft copolymers contemplated for use in the practice of the presentinvention are those wherein the polycationic species has grafted thereonat least one water-soluble polymer chain per chain of said polycationicspecies, up to a maximum of one grafted chain per repeat unit of saidpolycationic species. For example, when the molecular weight of thepolycationic species falls in the range of about 20,000, it willtypically have grafted thereon at least about 5 chains of saidwater-soluble polymer chain per chain of polycationic species; with inthe range of about 10-20 chains of said water-soluble polymer chain perchain of said polycationic species being the presently most preferredlevel of grafting. Those of skill in the art recognize that withpolycationic species having higher molecular weights, higher levels ofgrafting will be desirable, and that the above values for graftinglevels should be increased accordingly. Similarly, with respect to thewater-soluble component of invention graft copolymers, the use of highermolecular weight species will allow one to achieve substantially thesame result while grafting fewer (water-soluble) chains per chain ofpolycationic species.

Preparation of the graft copolymers of the present invention can becarried out employing chemical techniques known by those of skill in theart. For example, the free hydroxyl groups of the water-solublecomponent can be activated to render such groups susceptible tonucleophilic displacement. Thus, the free hydroxyl groups of thewater-soluble component can be subjected to esterification,etherification, amidation, urethane formation, and the like. Suchreactions involve the generation of such intermediates as carbonates,sulfonates, xanthates, epoxides, aliphatic aldehydes, carboxymethylazides, succinimidyl succinates, and the like. The activatedwater-soluble component can then be coupled to a suitable polycationicspecies, for example, by nucleophilic displacement.

Cell types contemplated for use in the practice of the present inventioninclude islets, fibroblasts, thyroid cells, parathyroid cells, adrenalcells, neuronal cells, dopamine secreting cells, hepatocytes, nervegrowth factor secreting cells, adrenaline/angiotensin secreting cells,norepinephrine/metencephalin secreting cells, human T-lymphoblastoidcells sensitive to the cytopathic effects of HIV, and the like.

Also included within the scope of the present invention are cells havinga modified cell surface which is substantially non-adhesive with respectto macromolecules encountered in physiological environments.

In accordance with another embodiment of the present invention, there isprovided a process to remove copolymer compositions contemplated for usein the practice of the present invention from cells treated as describedabove, said process comprising contacting such cells with an effectiveamount of an anionic species.

Anionic species contemplated for use in this embodiment of the presentinvention include monomeric or polymeric anions. Any soluble anionicspecies capable of reversing the association of polycationic specieswith negatively charged cell surface can be employed for this purpose.Presently preferred anionic species are polyanionic species, such as,for example, heparin, heparin sulfate, chondroitin sulfate, solublealginates (e.g., sodium alginate, potassium alginate, ammonium alginate,and the like), bovine serum albumin, hyaluronic acid, pectin,carageenan, oxidized cellulose, and the like.

"Contacting" of treated cells to remove invention copolymer therefrom iscarried out at room temperature for a time in the range of about 0.01 upto 1 hour or longer in physiological buffer solution containing anionicspecies at a sufficiently high ionic strength to reverse the associationof polycationic species with negatively charged cell surface.

An effective amount of anionic species to employ in accordance with thisembodiment of the present invention depends on the specific anionicspecies employed. Generally, the concentration of anionic speciesemployed will be sufficient to reverse polycation binding to cells ortissues, but not so high as to be toxic to the biological material beingtreated. Concentrations employed are typically in excess of the amountof anion actually needed to disrupt binding of polycation to cellsurface. Thus, for example, presently preferred treating solutionscontain about 2.5 Units/ml of heparin or 2 mg/ml of bovine serumalbumin.

In accordance with yet another embodiment of the present invention,there is provided a method to render cells non-immunogenic, said methodcomprising contacting said cells with an effective amount of acomposition comprising a polycationic species having water-solublepolymer chains grafted thereon.

"Contacting" of cells with graft copolymer compositions to render cellsnon-immunogenic is typically carried out as described above with respectto rendering cells non-adhesive.

The process of the present invention can be used for renderingnon-immunogenic any cell, tissue, organ, or system of organs, and thelike, that may be used for transplant or the like.

Also included within the scope of the present invention are cells havinga modified cell surface which is substantially non-immunogenic withrespect to mediators of immune response, e.g., biological macromoleculessuch as proteins, enzymes, and the like.

In accordance with another embodiment of the present invention, there isprovided a process to remove copolymer compositions contemplated for usein the practice of the present invention from cells treated as describedabove, said process comprising contacting treated cells with aneffective amount of an anionic species, as described above.

In accordance with still another embodiment of the present invention,there is provided a method to preserve cells and/or tissues which aresubjected to long periods of storage before being used for therapeuticapplications, said method comprising contacting said cells and/ortissues with an effective amount of a composition comprising apolycationic species having water-soluble polymer chains graftedthereon.

"Contacting" of cells and/or tissues with graft copolymer compositionsto preserve same is typically carried out as described above withrespect to rendering cells non-adhesive and/or non-immunogenic.

In accordance with a still further embodiment of the present invention,there is provided a method for associating water-soluble polymer with acell surface, said method comprising:

grafting water-soluble polymer onto a polycationic resin to produce acopolymer of said water-soluble polymer and said polycation, andthereafter

contacting said cell surface with an effective amount of said copolymer.

If desired, the copolymer can be substantially removed from the cellsurface employing the above-described removal process.

In accordance with a further embodiment of the present invention, thereis provided a method for the stabilization of liposomes havingnegatively charged surfaces, said method comprising contacting saidliposomes with an effective, stabilizing amount of a compositioncomprising a polycationic species having water-soluble polymer chainsgrafted thereon.

"Contacting" of liposomes for the stabilization thereof is carried outat room temperature for a time in the range of about 0.01 up to 1 houror longer in physiological buffer.

An effective amount of graft copolymer for use in this embodiment of thepresent invention is an amount sufficient to render such liposomesessentially non-detectable in vivo, thereby reducing uptake of theliposome by the reticuloendothelial system (and increasing liposomecirculation times in vivo). Suitable quantities of graft copolymer willrender the liposomes substantially non-adhesive to biological materials,while leaving the liposome intact, and without adversely affecting thefunction and/or activity of the contents thereof, if any. Typically, aconcentration of graft copolymer sufficient to neutralize the negativecharges on the surface of the liposome will be employed. Concentrationsin the range of at least about 0.05% of graft copolymer, with respect tothe concentration of the polycationic species used to treat the surfaceof said liposome will be employed; with concentrations of graftcopolymer in the range of about 0.1 up to 0.5% being presentlypreferred.

One can readily determine the stability of a liposome using a functionalassay, such as the following. In an in vitro setting, the stability ofliposome-encapsulated hemoglobin in an un-modified liposome could becompared to the stability of hemoglobin encapsulated in a liposomestabilized in accordance with the present invention (i.e., the result oftreating an un-modified liposome with a sufficient quantity of graftcopolymer described above to stabilize the liposome). The release ofhemoglobin into the surrounding buffer media over time would then beassayed, with an extended time-frame for release of hemoglobinindicating enhanced liposome stability.

In accordance with yet another embodiment of the present invention,there is provided a method for producing neural networks on a substrate,said method comprising:

masking that portion of said substrate which defines the desirednetwork,

rendering the unmasked portion of said substrate non-adhesive by theabove-described method of the invention,

removing the mask, then

allowing cells to spread and grow on said substrate, wherein cells growonly on the portion of the substrate which has not been treated withgraft copolymer.

Substrates contemplated for use in the above-described method includetissue culture substrates, such as collagen, tissue culture polystyrene,microporous dextran substrate, and the like.

Masking contemplated by the above-described method can be accomplishedin a variety of ways, such as, for example, by covering a portion of thesubstrate with an agent which does not serve as a substrate for cellgrowth (e.g., a piece of tape, or the like).

The masking agent employed can readily be removed by merely reversingthe process employed for applying the mask to the substrate.

Conditions required for cells to spread and grow on the substrate arestandard cell culture conditions.

The resulting neural networks can be used for a variety of purposes,such as, for example, for studying the transmission of nerve impulses,for connection between a nerve cell and an electrical circuit, and thelike.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES EXAMPLE I Synthesis of a Graft copolymer of Poly-L-Lysine andPolyethylene Glycol

Twenty grams (20 g) of PEG (molecular weight 10,000 g/mol, having thestructure HO-PEG-OH) were dried in a vacuum oven at 80° C. for 24 hoursand dissolved in 100 ml of methylene chloride that had been dried bymolecular sieves (4A). Then, 3.24 g of 1,1-carbonyldiimidazole (CDI, 5fold excess, to ensure the activation of 100% of PEG end groups) wereadded to the solution and stirred overnight at room temperature in anargon atmosphere. The CDI-activated PEG was then precipitated with anexcess of anhydrous diethyl ether and dried overnight under vacuum. Fivegrams (5 g) of the CDI-activated PEG were dissolved in 20 ml of 5 mMsodium borate buffer (pH 9). In order to prevent crosslinking ofpoly-L-lysine (PLL) with the 100% CDI-activated PEG, 50% of the PEG endgroups were inactivated by adding 30.2 μl of ethanolamine to the buffersolution and stirring for 4-6 hours at room temperature. This results ina mono-activated CDI-derivative of PEG, having the structure CDI-PEG-OH.Alternatively, a monomethoxy PEG could be used to avoid this partialdeactivation step, but monomethyl PEGs are presently availablecommercially only up to molecular weight 5000.

Following the above-described partial deactivation step, 50 mg of PLL(M.W. 20,100 g/mol) were added to the reaction mixture and stirred for24 hours at room temperature. The solution was then dialyzed for 24hours against deionized water and freeze dried to obtain a powder. Thisprocedure produced a PEG graft copolymer (PLL-PEG) having aconcentration of approximately 10-20 PEG chains per PLL chain.

EXAMPLE II Demonstration of Cell Binding Properties of PLL-PEG toFibroblasts: Effect on Cells in Suspension

The cell binding effects of PLL-PEG copolymer produced as described inExample I were tested on cultures of human foreskin fibroblasts (HFF).These cells are anchorage dependent and ordinarily die within 4 to 10hours if they do not adhere and spread on a surface. Thus, a flask ofconfluent HFF was harvested with trypsin-EDTA, then the resultant cellsin suspension were split into 6 batches, each containing approximately170,000 cells. Each batch was centrifuged to obtain cell pellets. Sixdifferent solutions were used for cell treatment:

(A) Fibroblast culture media: Dulbecco's modified Eagles' medium (D-MEM)containing 10% fetal bovine serum;

(B) 10 mM HEPES buffered saline (HBS), pH 7.4;

(C) HBS containing 0.1% (w/v) PLL;

(D) HBS containing 0.5% PEG (M.W. 10,000);

(E) HBS containing 0.1% PLL and 0.5% PEG; and

(F) HBS containing 0.3% PLL-PEG (based on PLL concentration).

All solutions were sterilized by filtration through 0.22 micron filtersprior to use. The cell pellets were resuspended in 2 ml of solutions A,B, C, D, E or F for approximately 10 minutes. The tubes were thencentrifuged (200×g for 5 minutes), the solutions aspirated and replacedwith fibroblast culture medium, and the cells plated onto culturedishes. The plated cells were observed periodically to verify adherenceand spreading. The cells were also stained with trypan blue (TB) to testviability. Table I summarizes the observations over 5 days following theseeding.

                                      TABLE I                                     __________________________________________________________________________            HFF TREATMENT SOLUTIONS                                                       A                                                                             (fibroblast                                                                         B                          F                                    TIME AFTER                                                                            culture                                                                             (buffered                                                                           C      D     E       (PLL-PEG                             SEEDING medium)                                                                             saline)                                                                             (PLL)  (PEG) (PLL + PEG)                                                                           copolymer)                           __________________________________________________________________________    1 hr    Normal                                                                              Normal                                                                              No     Normal                                                                              No      No                                           spreading                                                                           spreading                                                                           adherence;                                                                           spreading                                                                           adherence;                                                                            adherence;                                               Cell         Cell    No                                                       clumping     clumping                                                                              clumping                             % viability                                                                           95    95    0      90    0       80                                   24 hr   Normal                                                                              Normal                                                                              No     Normal                                                                              No      No                                           spreading                                                                           spreading                                                                           adherence;                                                                           spreading                                                                           adherence;                                                                            adherence;                                               Cell         Cell    No                                                       clumping     clumping                                                                              clumping                             % viability                                                                           100   100   0      95    0       70                                   48 hr   Confluent                                                                           Confluent                                                                           No     Confluent                                                                           No      No                                           monolayer                                                                           monolayer                                                                           adherence;                                                                           monolayer                                                                           adherence;                                                                            adherence;                                               Cell         Cell    No                                                       clumping     clumping                                                                              clumping                             % viability                                                                           100   100   0      95    0       60                                   120 hr  Confluent                                                                           Confluent                                                                           No     Confluent                                                                           No      No                                           monolayer                                                                           monolayer                                                                           adherence;                                                                           monolayer                                                                           adherence;                                                                            adherence;                                               Cell         Cell    No                                                       clumping;    clumping;                                                                             clumping;                                                Few spread   Few spread                                                                            Few spread                                               Cells        Cells   Cells                                % viability                                                                           100   100   <1     95    <3      60                                   __________________________________________________________________________

Treatments A, B, and D showed essentially the same results, with most ofthe HFF showing normal spreading and viability.

Free PLL was found to be toxic at the concentrations used (treatments Cand E). Essentially all cells subjected to treatments C and E took up TBand did not spread on the tissue culture substrate. The cells subjectedto treatments C and E also showed extensive aggregation.

Free PEG had no appreciable effect on cell function (treatment D). PEGalso had no appreciable ameliorating effect in conjunction with PLL(treatment E).

Incubation with the graft PLL-PEG copolymer of the present invention(treatment F) however, had a remarkable effect on the HFF. In starkcontrast to treatment with free PLL, treatment with the copolymerPLL-PEG (at 3 times higher concentration than used for treatments C andE) produced cells that showed no adherence to the substrate, noaggregation in suspension, but a high percent viability. This viabilitywas maintained for well over 24 hours with the HFF still in suspension.This behavior is quite unusual for anchorage dependent cells.

A distinct morphological difference in cells treated with PLL andPLL-PEG was evident. PLL treated cells in suspension showed a rough orragged surface while those treated with PLL-PEG copolymer of the presentinvention are smooth and spherical, much like freshly trypsinized cells.

These results indicate that treatment with the PLL-PEG copolymer of theinvention is noncytotoxic to HFF. In addition, interaction of thePEG-grafted polycation with the exterior of the cell prevents the cellfrom adhering to a substrate. Thus the cytotoxicity of PLL is markedlyreduced by PEG grafting.

Five days after the initial treatment, a few of the cells treated withPLL-PEG copolymer begin to show some spreading on the surface of theculture dish. This observation implies that the PLL-PEG copolymer mayeither have desorbed from the cell surfaces, or cell division may haveoccurred (which would dilute the concentration of PLL-PEG copolymer onthe cell membrane).

EXAMPLE III Assessment of Efficacy of PLL and PLL-PEG Treatments atVarious Dilutions

A similar experiment as outlined in Example II was conducted to test theeffects of PLL and PLL-PEG copolymer at various dilutions. Solutions Cand F were serially diluted with 10 mM HEPES buffered saline (HBS) to1/5, 1/25, and 1/125 of their original concentrations, and humanforeskin fibroblasts (HFF) incubated in these solutions for 10 minutes.Additional treatments included PEG 20M (a PEG composition having amolecular weight of about 20,000, comprised of two lower molecularweight PEGs (one having a MW ˜8,000 and the other having a MW of˜10,000) linked together by a hydrophobic, bifunctionalbisphenol-epichlorohydrin linker; available from Union Carbide, Danbury,Conn.) and PEG 20,000 (a substantially linear PEG having a molecularweight of ˜20,000; available from Fluka, Ronkonkoma, N.Y.) at 0.5% inHBS. A control treatment with fibroblast culture media was also run.Results are summarized in Table II, below.

In the Table, the following abbreviations are used:

"adh." for adhesion,

"aggreg." for aggregated, and

"subst." is the abbreviation for substrate.

P-0 refers to cells treated with 0.1% of PLL, and P-5, P-25 and P-125refer to cells treated with 1/5, 1/25, and 1/125 dilutions thereof,respectively. Similarly, G-0 refers to cells treated with 0.3% ofPLL-PEG copolymer, and G-5, G-25 and G-125 refer to cells treated with1/5, 1/25, and 1/125 dilutions thereof, respectively.

                                      TABLE II                                    __________________________________________________________________________           HFF TREATMENT SOLUTIONS                                                TIME   Control                                                                AFTER  No  20M 20,000                                                                            PLL treatment           PLL-PEG copolymer treatment        SEEDING                                                                              PEG PEG PEG P-0   P-5   P-25  P-125 G-0   G-5  G-25                                                                              G-125               __________________________________________________________________________    0 hr   Minor adh.  Clumped                                                                             Clumped                                                                             Clumped                                                                             Clumped;                                                                            No adh.                                                                             No adh.                                                                            Minor                                                                             Minor                      to                            adh. to          adh.                                                                              adh. to                    substr.                       subst.           subst.                                                                            subst.              3 hr   100%        Clumped;                                                                            Clumped;                                                                            Clumped;                                                                            Clumped;                                                                            No    50%  75% 100%                       adh.        aggreg.;                                                                            aggreg.;                                                                            5-10% 5-10% adh.  adh. adh.                                                                              adh. to                    to          no adh.                                                                             no adh.                                                                             adhered                                                                             adhered          subst.                                                                            subst.                     subst.      to subst.                                                                           to subst.                                                                           to subst.                                                                           to subst.                                24 hr  100%        No adh;                                                                             No adh;                                                                             Clumped;                                                                            Clumped;                                                                            No    ˜60%                                                                         ˜80%                                                                        ˜100%                adhesion    Extensive                                                                           Extensive                                                                           ˜10% adh.                                                                     ˜10% adh.                                                                     adh.; adh.;                                                                              adh.                                                                              adhesion                               clumping                                                                            clumping                                                                            to subst.                                                                           to subst.                                                                           no                                                                            clumping                           %      100%        <10%  <10%  <10%  <10%  >70%  >70% >70%                                                                              >70%                viability                                                                     __________________________________________________________________________

Observation of the cells immediately after seeding showed all PLLtreatments (abbreviated P) to cause clumping of cells. A small number ofcells showed adherence in the P-125 treatment. The graft copolymer(PLL-PEG, abbreviated G) treatment showed a decrease in efficacy at thelower concentrations. At dilutions of 25 and 125 (G-25 and G-125),adherence of cells was noted, though not quantified. Treatments with thePEG 20M and PEG 20,000 showed no appreciable difference from thecontrol.

Three hours following the initial seeding, the following observationswere made. The PLL treated cells P-0 (0.1% PLL) and P-5 were clumped andaggregated, with none of the cells showing adherence to the substrate.P-25 and P-125 also showed clumping, but approximately 5-10% of cellsadhered to the substrate, indicating PLL cytotoxicity at very lowconcentrations.

Cells treated with PLL-PEG showed an increased adhering tendency withincreasing dilutions. G-0 (0.3% PLL-PEG) showed no adhesion andindividual free-floating cells. G-5, G-25, and G-125 showedapproximately 50%, 75% and 100% adherence, respectively, at 3 hours.G-125 was very similar to the PEGs and the control.

After 24 hours, P-0 and P-5 showed no adherence to substrate, andextensive clumping. P-25 and P-125 also showed clumping, butapproximately 10% of the cells were adhered to the substrate, indicatinga lower level of toxicity for P-25 and P-125, compared to the higherconcentrations used in samples P-0 and P-5.

After 24 hours, G-0 showed no adherence to substrate and no clumping;while G-5, G-25 and G-125 showed increasing levels of adherence ofapproximately 60%, 80% and 100%, respectively. The PEG treatments andthe control were also 100% adhered.

TB staining at 24 hours showed all PLL treatments to have less than 10%viability, while the treatments with PLL-PEG copolymer showed aviability of greater than 70%. Thus the attachment of PEG to PLLsubstantially alleviates the PLL toxicity; this effect is apparent atvery low concentrations (P-125=0.0008% PLL; G-125=0.0024%).

EXAMPLE IV Effect of PLL and PLL-PEG on Confluent Monolayers ofFibroblasts

In order to assess, in a more realistic (although in vitro situation),the effects of PLL and PLL-PEG copolymer of the invention on cells whichwould normally be present in a flattened spread morphology (and not in arounded morphology), confluent monolayers of HFF were treated withsolutions P-0, P-5, G-0, G-5, PEG 20M, and a control (fibroblast culturemedium). The cells were exposed to these solutions for 10 minutes,followed by a rinse with HBS, then fibroblast culture medium wasreturned to the culture dishes.

Short-term observation 15 minutes after treatment showed the P-0 treatedcells sloughing off the culture substrate, with approximately 90% of allcells in suspension at 20 minutes.

About 2-5% of cells treated with P-5 were detached from the surfacewithin the same 15 minute period.

HFF treated with solutions G-0, G-5 and PEG 20M showed no appreciabledifference from the control cells.

These results indicate that PLL (at 0.1%) is clearly toxic to HFF, whilesimilar concentrations of PLL modified with PEG show no harmful effectsto confluent monolayers of cells. It is noteworthy that the P-5treatment showed only mild toxicity to spread, confluent fibroblasts,indicating that they may be less susceptible to toxic macromolecules inthis state rather than in suspension.

EXAMPLE V Reversal of PLL-PEG Binding to Cells with Polyanions

It was possible, by addition of heparin sulfate or chondroitin sulfate,to reverse the effect of PLL and PLL-PEG on HFF. Thus, addition of 2.5U/ml of heparin to the fibroblast culture medium soon after treatmentwith PLL caused disaggregation of the HFF clumps and resulted in cellsthat were able to adhere to tissue culture substrates. If, however, theaddition of heparin was postponed until several hours after the PLLtreatment, reversibility was not possible because the cells hadsuccumbed to PLL toxicity.

This however, was not the case with the PLL-PEG copolymer if the presentinvention. The nonadhesive, nonaggregating nature conferred upon thefibroblasts by treatment with PLL-PEG copolymer was found to bereversible at least 48 hours after the initial treatment, clearlyindicating that these anchorage dependent cells were still alive,despite the fact that they were not adhered to a substrate.

EXAMPLE VI Resistance of PLL-PEG Treated Cells to specific Antibodies asIndicators of Conferred Immune Protection

Fibroblasts have receptors for the protein vitronectin on theirsurfaces. Vitronectin is a cell adhesion molecule (CAM). This receptor(called αV-β3) can be targeted with an antibody, anti αV-β3, a rabbitpolyclonal. A fluorescently conjugated secondary antibody to anti αV-β3(e.g., rhodamine conjugated anti IgG, goat anti-rabbit) would permit thevisualization of these receptors on the cell surface.

Untreated HFF, PLL treated HFF, and PLL-PEG treated HFF were incubatedwith anti αV-β3 polyclonal antibody, followed by incubation with thesecondary antibody, then observed at the appropriate excitationwavelengths under a microscope. It was observed that the untreated andPLL treated cells showed strong fluorescence, while the PLL-PEG treatedcells fluoresced at a much lower level. This observation indicates thatthe approach of the antibody to the cell is hindered by the presence ofPEG.

PLL by itself was found not to affect the receptor-ligand interaction.

Based on the above-described experiments, it is likely that theprevention of protein binding to these cells will render themimmunologically unrecognizable.

EXAMPLE VII Transplantation of PLL-PEG Treated Allogeneic Islets in Ratsas a Model for Immunoprotectivity

Rat islets were isolated employing techniques known in the art [see, forexample, Lacy and Kostianovsky in Diabetes 16:35 (1967)]. The isolatedislets were treated with 0.3% PLL-PEG as described above (see ExampleII), and transplanted by injection into the peritoneal cavity ofdiabetic rats. Diabetes was induced by treatment with streptozotocin.Controls were injected with untreated rat islets. Blood glucose levelsof these rats were monitored at first on an hourly basis, and then on adaily basis for several weeks. It was found that the control rats had areversal of diabetes (indicated by normal glucose levels) for 3-4 daysfollowing which the graft failed due to rejection. On the other hand,the rats injected with the PLL-PEG copolymer treated islets showed acontinuous reversal of diabetes for several weeks, indicating that thetreatment of these cells with PLL-PEG copolymer was effective inimmunoprotecting the islets.

EXAMPLE VIII Crosslinkable Graft Copolymers

A variation on the above theme for the surface treatment of cells is onein which the PLL-PEG graft copolymer has on its structure polymerizablegroups such as the acrylate group. The presence of this group on thegraft copolymer facilitates polymerization or crosslinking following theabsorption of the copolymer onto the cell surface through ionicinteractions. The resultant covalently crosslinked network issignificantly more stable than the ionically attached graft copolymer.Thus the immunoprotective properties conferred upon the cell byabsorption of PLL-PEG on its surface are no longer transient as may beexpected through an ionic interaction, but are permanent due to theformation of intermolecular and intramolecular covalent crosslinksformed with the PLL-PEG.

Synthesis of these polymerizable copolymers could have two possiblestrategies. One involves the synthesis of a PEG that isheterobifunctional, i.e., one end is functionalized with CDI(1,1-carbonyldiimidazole; or other electrophilic derivative) and theother with acryloyl chloride (the reaction of PEG with acryloyl chlorideis described below). This technique allows the synthesis of a PLL-PEGgraft copolymer in which the free end of PEG contains a polymerizabledouble bond. The second strategy involves the preparation of PLL-PEG asdescribed above, and the subsequent reaction of the copolymer withacryloyl chloride to add polymerizable groups. In this case the additionof polymerizable groups to the copolymer is nonspecific, i.e., thesubstitution occurs on the free end of the PEG as well as on the amineson polylysine.

The reaction of PEG with acryloyl chloride proceeds to completion inabout 24 hours when carried out at 50° C. For example, mono-CDIfunctionalized PEG (i.e., CDI-PEG-OH, prepared as described in ExampleI) was reacted with an equimolar amount of acryloyl chloride in drydichloromethane solvent. The reaction was carried out in a round-bottomflask under an inert atmosphere at constant reflux for 24 hours. Theresulting product was purified by precipitation with diethyl ether, thendried in a vacuum oven.

Alternatively, PLL-PEG could be treated with acryloyl chloride. In thissituation, acrylate substitution would occur on both the PEG chains andthe PLL backbone (via the amine groups thereof).

Photopolymerization is the method of choice for covalent crosslinking ofthe graft copolymer following attachment to the cell surface. Followingattachment of the graft copolymer to the cell surface the treated cellsare transferred to a physiological buffer solution containing ethyleosin (EE, 0.1 μM to 0.1 mM), triethanolamine (TEA, 0.1 mM to 0.1M), andoptionally a comonomer (e.g. 1-vinyl 2-pyrrolidinone (VP) at aconcentration in the range of about 0.001 to 1.0%, when used). Thissolution containing islets is well mixed and exposed to a mercury lamp(100 watt) with a bandpass filter (500-560 nm) for approximately 3minutes. This causes crosslinking of the copolymer on the surface of thecell resulting in the immunoprotective layer. The cells are thentransferred to culture.

An alternative technique involves the incubation of the copolymertreated cells with a solution of EE (0.1 μM to 0.1 mM) in physiologicalbuffer for approximately two minutes. In this step the EE complexes withthe positively charged polycation on the cell surface. After rinsing inbuffer the cells are transferred to a physiological buffer solutioncontaining TEA (conc. as above), and a comonomer e.g. VP (optional).This solution containing islets is well mixed, polymerized as before,and transferred to culture.

EXAMPLE IX PLL-PEG Solutions in Organ Preservation Media

As noted above, PEG 20M has been used in the preservation of organs. Thebasis of its activity, though not clearly understood, is believed to bethe binding of PEG to cell surface molecules through nonspecifichydrophobic interactions. The PLL-PEG copolymer of the presentinvention, however, interacts directly through ionic interactions withcell-surface moieties bearing a negative charge. Thus, tissues andorgans may be flushed with a solution containing the PLL-PEG copolymerprior to transplantation to, in effect, `coat` the tissues with PEG,thereby providing an immunoprotective and organ-protective effect.

EXAMPLE X Stabilization of Liposomes with PLL-PEG for Longer CirculationTimes and Increased Biocompatibility

Lipid vesicles or liposomes have been investigated extensively assystems for drug delivery (Gregoriadis, 1987). The commonly usedphospholipids that comprise liposomes, such as phosphatidyl choline,phosphatidyl serine, dilaurylphosphatidic acid, and phosphatidylglycerolare negatively charged at physiological pH. The interaction ofpolycations such as PLL with the negatively charged phospholipids hasbeen studied quite extensively with regard to conformational changesinduced in PLL and consequent stability [Fukushima et al., BiophysicalChemistry 34:83 (1989); Houbre et al., Biophysical Chemistry 30:245(1988)]. Stability of liposomes in physiological conditions is a majorfocus of researchers investigating drug delivery. Although PLL may beused to stabilize lysosomes in vitro, PLL coated liposomes in vivo arelikely to be rapidly overgrown or ingested by macrophages due to theadhesive nature of PLL, thus making them ineffective for the controlledrelease of drugs. In addition, liposomes may also be destroyed by uptakeby the reticuloendothelial system. The addition of the graft copolymersof the present invention to the surface of the liposome is likely toprevent this uptake.

The replacement of PLL by the PLL-PEG copolymer of the presentinvention, however, promises to provide a liposome that is stable notonly due to interactions between negatively charged phospholipid andpositively charged PLL, but also because the PLL-PEG copolymer willprevent interactions with proteins, and therefore prevent interactionswith cells such as macrophages. This should result in liposomes withlong circulation times which can therefore deliver drugs in a controlledfashion.

EXAMPLE XI Patterned Surfaces for Neural Networks

Investigators in neurology have tried to generate in vitro networks ofneurons on culture dishes. A problem has been to generate patternedsurfaces that are preferentially adherent to cells in order to design`biological circuits.` By creating a mask of the pattern desired, andapplying it to the culture substrate, followed by treatment of thesurface with PLL-PEG copolymer, one can selectively leave the desiredpattern adhesive to cells, while the rest of the available surface isrendered nonadhesive to cells.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

That which is claimed is:
 1. A method to render cells non-adhesive, saidmethod comprising directly contacting a surface of said cells with aneffective amount of a composition comprising a polycationic specieshaving water-soluble polymer chains grafted thereon.
 2. A methodaccording to claim 1 wherein said water-soluble polymer is selected frompolyethylene glycol (PEG), polyvinyl alcohol (PVA), poly(hydroxyethylmethacrylate) (pHEMA), polyacrylic acid (PAA), poly(acrylamide), poly(vinyl pyrrolidone) (PVP), poly(ethyl oxazoline) (PEOX),polysaccharides, or copolymers of any two or more thereof.
 3. A methodaccording to claim 1 wherein said polycationic species has graftedthereon at least one water-soluble polymer chain per chain of saidpolycationic species.
 4. A method according to claim 3 wherein saidwater-soluble polymer is polyethylene glycol.
 5. A method according toclaim 1 wherein either the water-soluble polymer, or the polycationicspecies, or both contain at least one functional group which issusceptible to free radical polymerization.
 6. A method according toclaim 5 wherein said composition is further subjected to free radicalpolymerization conditions.
 7. A method according to claim 1 wherein saidpolycationic species is selected from one or more of:polyethyleneimine,polyallylamine, polyetheramine, polyvinylpyridine, polysaccharideshaving a positively charged functionality thereon, polyamino acidsselected from:poly-L-histidine, poly-im-benzyl-L-histidine,poly-D-lysine, poly-DL-lysine, poly-L-lysine, poly-ε-CBZ-D-lysine,poly-ε-CBZ-DL-lysine, poly-ε-CBZ-L-lysine, poly-DL-ornithine,poly-L-ornithine, poly-δ-CBZ-DL-ornithine, poly-L-arginine,poly-DL-alanine-poly-L-lysine; poly(-L-histidine, L-glutamicacid)-poly-DL-alanine-poly-L-lysine; poly(L-phenylalanine, L-glutamicacid)-poly-DL-alanine-poly-L-lysine; or poly(L-tyrosine, L-glutamicacid)-poly-DL-alanine-poly-L-lysine; or random copolymers of:L-argininewith tryptophan, tyrosine, or serine; D-glutamic acid with D-lysine; orL-glutamic acid with lysine, ornithine, or mixtures thereof.
 8. A methodaccording to claim 1 wherein said polycationic species is selected frompolylysine or polyornithine.
 9. A method according to claim 1 whereinsaid cells to be rendered non-adhesive are selected from islets, thyroidcells, adrenal cells, dopamine secreting cells, hepatocytes, or humanT-lymphoblastoid cells sensitive to the cytopathic effects of HIV. 10.The cells produced by the method of claim
 1. 11. A method to rendercells non-immunogenic, said method comprising directly contacting asurface of said cells with a composition comprising a polycationicspecies having water-soluble polymer chains grafted thereon.
 12. Amethod according to claim 11 wherein said polycationic species hasgrafted thereon at least one water-soluble polymer chain per chain ofsaid polycationic species.
 13. A method according to claim 12 whereinsaid water-soluble polymer is polyethylene glycol.
 14. A methodaccording to claim 11 wherein said cells to be rendered non-immunogenicare selected from islets, thyroid cells, adrenal cells, dopaminesecreting cells, hepatocytes, or human T-lymphoblastoid cells sensitiveto the cytopathic effects of HIV.
 15. The cells produced by the methodof claim
 11. 16. A method for associating water-soluble polymer with acell surface, said method comprising:grafting water-soluble polymer ontoa polycationic resin to produce a copolymer, and thereafter directlycontacting said cell surface with an effective amount of said copolymer.17. A method according to claim 16 wherein said copolymer comprises apolycation having grafted thereon at least one water-soluble polymerchain per chain of said polycationic resin.
 18. Cells having a modifiedcell surface which is non-adhesive with respect to mediators of immuneresponse, wherein the surface of said cells has been modified bydirectly contacting said surface with a composition comprising apolycationic species having water-soluble polymer chains graftedthereon.